Auditory and vestibular function are dependent of the formation of a functional inner ear. While there are multiple components for both of these systems, this laboratory focuses on the development of the sensory epithelia, which contain mechanosensory hair cells and associated cells called supporting cells and on the innervation of those hair cells by neurons from the VIIIth (acousticovestibular) cranial nerve. All three of these cell types are derived from the otocyst, a placodal structure that forms adjacent to the hindbrain early in development. Identifying the factors that specify each of these cell types and then direct their assembly into functional units is a key goal of the Section on Developmental Neuroscience. During the previous year, different members of the laboratory have examined several different aspects of these developmental processes. Spiral ganglion neurons (SGNs) are afferent fibers that act as the sole relay between the auditory sensory epithelium of the cochlea and the brain. Historically, SGNs were divided into two classes, Type Is which form synapses on inner hair cells and Type IIs which form synapses on outer hair cells. However, considering that SGNs must be able to interpret the full complexity of auditory perception, it seemed unlikely that all SGN neurons would be the same. Through several screening studies we identified a transcription factor, Pou4f1, that is expressed in a subset of Type I SGNs. To determine whether those SGNs might share any morphological or functional traits, we used a combination of genetic sparse-labeling with immunofluorescence to characterize Pou4f1-positive SGNs. We found that nearly 100% of Pou4f1-positive SGNs form synapses in the same sides of inner hair cells, suggesting that they probably comprise a subset of SGNs that are characterized by low spontaneous rates of firing. To examine the potential physiological roles of these fibers we generated SGN-specific knock-outs of Pou4f1. Hearing in these animals, based on auditory brain stem recordings (ABR) were normal. However, in collaboration with the laboratory of Tobias Moser, we were able to demonstrate that deletion of Pou4f1 leads to several significant changes in function of the synapses between inner hair cells and SGNs. In particular, changes in the size and density of synaptic ribbons in the hair cells was altered. This study represented the first demonstration of the ability of a post-synaptic SGN to alter presynaptic structure in the hair cells. Ongoing studies in the laboratory will continue to examine the effects of Pou4f1 as well as to search for additional markers of diversity within the SGN population. The sensory epithelium of the mammalian cochlea, also called the organ of Corti, has a very unique cellular structure that is comprised of two different types of supporting cells along with at least 6 different types of surrounding supporting cells. One of the more striking aspects of this structure is its distinct separation into medial and lateral regions which each contain different types of hair cells and supporting cells. The medial region contains a single row of inner hair cells and two supporting cell types, inner phalangeal cells and border cells, while the lateral region contains three rows of outer hair cells and three types of supporting cells, inner pillar cells, outer pillar cells and Deiters cells. How the epithelium becomes partitioned into these distinct medial and lateral domains is unclear. Based on its pattern of expression and known role in other systems, we suspected that Glycogen synthase kinase 3 beta (GSK3b), a signaling molecule that plays a role in patterning in a number of other systems, might act in a similar fasion in the cochlea. In fact, following inhibition of GSK3b we observed a striking change in the pattern of the cochlea. While the total number of hair cells was largely unchanged, there was an increase in the number of inner hair cells and a proportional decrease in the number of outer hair cells. Subsequent experiments demonstrated that the effects of GSK3b are modulated through two different signaling pathways, the Bmp4 and notch pathways. In fact, many of the changes in cellular patterning that occur following inhibition of GSK3b can be prevented by increasing the amount of Bmp4. These results provide the first demonstration of a signaling pathway that regulates medial and lateral regions of the cochlear duct. A significant challenge in understanding cellular diversity within the auditory and vestibular systems is the presence of a relatively high number of cell types combined with a relatively low number of any given type of cell. Over the last several years, we have taken advantage of the development of new technologies to profile RNA expression in single cells. While much of this work has focused on the cochlea, we have also examined cellular diversity in the utricle, a vestibular organ involved in the perception of acceleration. The results of an initial single cell RNA sequencing experiment allowed us to identify new markers for both types of hair cells (Type I and Type II) within the utricle. Among these markers were genes that did not turn on until cells had become committed to become either Type I or Type II utricular hair cells. Using these markers we were able to study the development of different cell types within the utricle. One of the more significant findings was that over 95% of all Type I hair cells are generated prior to birth in the mouse. This disproved a longstanding theory that Type I hair cells actually arose as a result of a conversion of Type II hair cells into Type I hair cells. Our results demonstrated that this theory is incorrect and instead Type I hair cells actually form first. Ongoing single cell studies are using new technologies to provide greater details regarding cell types and developmental processes in both the cochlea and utricle. Finally, we have happily shared our expertise in single cell isolation and analysis with other laboratories at the NIDCD, the NIH and in the extramural community. This has resulted in the publication of several manuscripts over the past year with members of the Section on Developmental Neuroscience as authors.